- American Society of Plant Biologists
In a letter addressed to Joseph Hooker in 1879, Charles Darwin referred to the rapid development and diversification of the flowering plants in recent geological times as an “abominable mystery.” The mystery of the origin of angiosperms, the development of floral characters, and the regulation of flowering were the topics of the 14th Penn State Symposium in Plant Physiology, organized by Claude dePamphilis, Teh-Hui Kao, Hong Ma, and Andrew Stephenson and held in State College, Pennsylvania, May 16 to 18, 2002. The program brought together researchers studying all aspects of plant reproduction and using a wide range of approaches, from evolutionary, physiological, and ecological analyses to molecular genetic investigations. This report presents a samp-ling of the many excellent presentations given.
EVOLUTION OF ANGIOSPERMS AND FLORAL CHARACTERS
Phylogenetic analysis of the flowering plants is a tremendously difficult task because of the explosive radiation of early angiosperms, which resulted in enormous diversity of floral characters, and the great divergence between angiosperms and gym-nosperms, among other factors. Pamela Soltis (University of Florida, Gainesville) gave a summary of angiosperm phylogeny and discussed patterns and mechanisms of floral evolution from a phylogenetic perspective. Recent work by the Soltises and their colleagues, based on extensive DNA analysis (Qiu et al., 1999; Soltis et al., 2000), has shown that the earliest angiosperms (basal branches of the angiosperm phylogenetic tree) are represented by Amborellaceae, Nymphaeaceae, and another clade that contains Illiciaceae, Schisandraceae, Trimeniaceae, and Austrobaileyaceae (the ITA clade) (Figure 1) . The basal branches are sister groups to all of the remaining angiosperms, which make up a strongly supported single large clade (euangiosperms).
Basal Angiosperm Phylogeny.
Major clades of basal angiosperms according to Qiu et al. (1999) and Soltis et al. (2000). Photographs show flowers of a representative genus within each group.
Interestingly, despite the importance and number of monocot species, their placement in angiosperm phylogeny is unresolved. The floral morphology of the basal branches and the magnoliids is extremely labile, suggesting that a number of changes and reversions of floral forms occurred repeatedly during angiosperm evolution. The floral diversity of these groups also suggests that there were numerous “experiments” in floral morphology in the basal groups that were unsuccessful and not retained through angiosperm evolution. There also are examples of great diversity within a clade (e.g., magnoliids) and great conservation within clades (e.g., grasses, orchids, and legumes).
Douglas Soltis (University of Florida) spoke in more detail about the evolution of floral characters. Many patterns of floral evolution among the angiosperms can be revealed by detailed examinations of four principal floral characters: merism (the number and growth patterns of floral meristems), phyllotaxis (spiral versus whorled arrangement of organs), perianth differentiation, and gynoecial diversification (ovary position). There is a huge amount of diversity in these characters across the angiosperms, but certain patterns emerge. For example, the core eudicots have apparently become “locked” in pentamery (meristems producing whorls of five organs), but merism is labile in the basal angiosperms, suggesting that there were many early experiments (e.g., in dimery and trimery). Overall, the analyses suggest that floral evolution has not occurred along simple linear pathways but rather is extremely dynamic and has involved multiple origins and evolutionary reversals of the various floral characters.
THE FLORAL GENOME PROJECT
Claude dePamphilis (Pennsylvania State University, State College) gave an overview of the Floral Genome Project, an ambitious undertaking linking phylogenetic, genomic, and developmental perspectives on plant reproduction to learn more about the origin, conservation, and diversification of the genetic architecture of flowers. The project, funded by the National Science Foundation from October 2001 through 2006, involves principal investigators at Penn State (dePamphilis, Hong Ma, Webb Miller, and John Carlson), Cornell University (Steven Tanksley and Jeff Doyle), the University of Florida (Pamela and Douglas Soltis), the University of Alabama (David Oppenheimer), and foreign collaborators Victor Albert (Oslo, Norway), Steve Farris (Stockholm, Sweden), Dawn Field (Oxford, United Kingdom), Michael Frohlich (London, United Kingdom), and Gunter Theissen (Jena, Germany).
The group has identified 15 representative species from gymnosperms, basal angiosperms, monocots, and core eudicots and will collect >100,000 ESTs involved in early flower development, obtain finished cDNA sequences of many of the genes from each species, and examine spatial and temporal patterns of gene expression. The project will generate a comparative data set of expression patterns for a large number of genes involved in flower development across diverse angiosperms as well as test standing hypotheses regarding the origin of the floral developmental program. Progress can be followed and more information obtained from the project World Wide Web site (http://fgp.bio.psu.edu).
EVOLUTION AND FUNCTION OF FLORAL REGULATORS
Floral development in angiosperms is dependent on the ABC genes, which work together to determine flower structure (Riechmann and Meyerowitz, 1997; Theissen et al., 2000). Most of the ABC genes encode MADS domain regulatory proteins. Gunter Theissen (University of Jena, Germany) described studies on the molecular evolution of MADS box floral homeotic genes. ABC MADS box genes are ubiquitous among the angiosperms, but they have not been found in ferns and other “lower” plants. Theissen and colleagues have investigated the model gymnosperm Gnetum gnemon and found that it has orthologs of the angiosperm B and C genes.
This raises the question of functionality, because angiosperm B and C genes specify the identity of petals, stamens, and carpels, yet gymnosperms lack a differentiated perianth (including petals) and have no stamens or carpels. Gene expression analysis showed that GGM2 (a Gnetum B homolog) is expressed only in male organs, whereas GGM3 (a Gnetum C homolog) is expressed in both male and female organs (Theissen et al., 2000). The working hypothesis is that sex determination of the ancestral seed plants was controlled by the differential expression of B and C genes; expression of both B and C led to the development of male organs, C expression alone led to the development of female organs, and the lack of both B and C expression led to vegetative organ development. This hypothesis has been tested by experiments that include ectopic expression of the Gnetum genes in floral mutants of Arabidopsis (Winter et al., 2002).
MADS box genes make up large gene families. In Arabidopsis, for example, this family has at least 100 members. The likely functional redundancy of many of these genes complicates functional analysis. Richard Immink (Plant Research International, Wageningen, The Netherlands) de-scribed efforts aimed at elucidating the function of MADS domain proteins through yeast two-hybrid protein–protein interaction assays. Immink and colleagues used two-hybrid screening between the Arabidopsis SEP3 MADS domain protein and 23 known MADS domain proteins from petunia to show that SEP3 is the functional ortholog of the petunia FBP2 MADS domain protein (Immink et al., 2002). This work demonstrates that protein–protein interaction mapping can be used to identify orthologs of MADS domain proteins from nonrelated species.
EVOLUTION OF THE TRIPLOID ENDOSPERM
The phenomenon of double fertilization in angiosperms was recognized as early as the late 1800s. For example, light microscopy images showing clear evidence of the fusion of one sperm cell with the egg cell to form the diploid zygote and a second sperm cell with the central cell to form the endosperm were published by Nawaschin (1898) and Guignard (1899). Despite detailed knowledge of these events for >100 years, relatively little is known about the evolution and genetic control of angiosperm fertilization and endosperm development.
William Friedman (University of Colorado, Boulder) discussed the evolution of fertilization in the angiosperms and the evidence that triploid endosperm may have arisen not with the earliest angiosperms, as has been widely supposed for the last century, but with later evolving lineages of core euangiosperms. The evolution of the triploid endosperm is a key event in angiosperm evolution and is not without importance to humans, because approx-imately two-thirds of human caloric intake worldwide is plant endosperm, such as rice.
Until recently, magnolia-like plants were widely considered to be models for the early angiosperm flower. The identification of the Amborellaceae, Nymphaeaceae, and the ITA clade as the earliest basal angiosperm lineages (Mathews and Donoghue, 1999; Qiu et al., 1999) has led to a reexamination of early angiosperm floral morphology and reproductive biology. The water lily Nuphar (Nymphaeaceae) produces a four-celled female gametophyte, which could develop from a seven-celled gametophyte after fusion of the two central cell nuclei (polar nuclei) and programmed cell death of three antipodal cells or from a “true” four-celled gametophyte. In the latter case, double fertilization would lead to the production of a diploid endosperm.
Williams and Friedman (2002) documented double fertilization in Nuphar and showed that the genus Nuphar (and probably other genera within the Nymphaeales and the ITA clade) contains true four-celled female gametophytes with a haploid central cell that produces a diploid endosperm after fertilization. However, Amborella, the only extant genus in the basal Amborellaceae clade, contains a seven-celled female gametophyte with a diploid central cell that is inferred to produce a triploid endosperm (double fertilization has not been documented in this taxon). Thus, it remains unresolved whether the earliest angiosperms had a diploid or a triploid endosperm.
The difference between a four-celled and a seven-celled female gametophyte lies in the early development of the female gametophyte. The first (megaspore) nucleus either (1) divides and the daughter nuclei migrate to opposite poles, where they undergo two more replications and divisions, followed by cellularization and fusion of the two central cell nuclei to produce a seven-celled gametophyte, or (2) divides and the daughter nuclei remain together at the micropylar end, where they undergo just one additional replication and division to produce a four-celled gametophyte with only one haploid nucleus in the central cell. A key step, then, is whether or not there is migration of the first two daughter nuclei. Further analysis of gametophyte ontogeny in the basal angiosperm lineages should provide more information regarding these critical events in angiosperm reproduction.
POLLEN TUBE GROWTH
Zhenbiao Yang (University of California, Riverside) is using pollen tube growth as a model for cell polarity development. Pollen tubes are basically single cells that elongate by polarized tip growth, which requires the establishment of an apical plasma membrane domain and the regulation of polar exocytosis in this region. Yang focused on the activity of ROP1, a member of the ROP family of plant-specific Rho-related small GTPases that is expressed specifically in pollen and pollen tubes and localizes preferentially to the tip growth domain. The group constructed dominant negative (locked in the GDP form) and constitutively active (locked in the GTP form) mutants of ROP1 and showed that overexpression of either of these mutant forms under the control of a pollen-specific promoter caused the inhibition of pollen tube elongation (Li et al., 1999). The dominant negative mutant ROP1 caused an overall inhibition of growth, whereas the constitutively active mutant caused a loss of growth polarity, suggesting that ROP1 is involved in polarity control.
Yang hypothesized that ROP1 activity defines the tip growth domain and controls polar tip growth, because inhibition of ROP1 activity inhibited growth, low levels of activity promoted polar growth, and a dramatic increase in activity was associated with depolarized tip growth. Pollen tube growth oscillates, and it has been shown that tip-localized F-actin and calcium gradients also oscillate during tip growth. Yang and colleagues have shown that the dynamics of tip-localized F-actin and calcium oscillations are dependent on ROP1 activity (Li et al., 1999; Fu et al., 2001). They also have shown that ROP1 signaling is critical for pollination. They are now working on identifying and characterizing ROP-Interactive CRIB motif (RIC) proteins using yeast two-hybrid screens and homology searching; they have identified 11 RIC proteins in Arabidopsis (Wu et al., 2001). Two of these, RIC3 and RIC4, may be ROP1 effectors that coordinately regulate calcium and F-actin dynamics in pollen tip growth.
FERTILIZATION AND FEMALE GAMETOPHYTE DEVELOPMENT
Ueli Grossniklaus (University of Zürich, Switzerland) spoke about the search for genes involved in female gametophyte development using enhancer detection. This technique uses a β-glucuronidase reporter gene under the control of a minimal promoter. If the T-DNA or transposon-based enhancer detector inserts close to cis-regulatory elements, β-glucuronidase is expressed in a spatial and temporal pattern similar to that of a nearby gene. This technique is particularly useful for the study of gametogenesis, because it allows for the detection of gene expression in one or just a few cells.
Grossniklaus and colleagues have identified many lines that show expression patterns covering all stages of gametophytic development. One of the insertional mutants isolated is feronia, a semisterile mutant in which half of the ovules do not initiate seed development. Megagametogenesis appears normal until fertilization, but then there is an arrest of growth after pollen tube arrival. The pollen tube enters the micropyle and keeps growing, producing a mass of abnormal material at the micropylar end of the megagametophyte. During normal pollination, the pollen tube enters one of the synergid cells, which degenerates in the process, and then ruptures to release the two sperm. The feronia mutation is hypothesized to interfere with synergid function, which is consistent with the phenotype and the expression pattern of the gene disrupted in the mutant.
Grossniklaus described another very interesting project in maize to investigate polyspermy in plants, which is the fertilization of one egg cell by more than one sperm. Polyspermy is strictly blocked in animals, ensuring that only one sperm fertilizes an egg. Genetic experiments by Grossniklaus and colleagues have shown that the block to polyspermy in plants is not as complete as it is in animals. The low, but detectable, rate of polyspermy in plants may be one of the mechanisms that allows for polyploidy, which occurs far more often in plants than in animals.
MATING STRATEGY
The evolution of self-pollinating versus outcrossing plant species is a complex and fascinating facet of plant reproduction. Interestingly, the transition to selfing is very common among flowering plants, and there are many examples of outcrossing and selfing sister taxa. John H. Willis (Duke University, Durham, North Carolina) and colleagues are exploring the genetic basis of phenotypic differences between sister taxa in the genus Mimulus (Scrophulariaceae or foxglove family) that have divergent mating systems. The Mimulus species are primarily bee pollinated and have classic bee flowers, with a “landing pad” formed by the lower lip of the corolla, a patterned “runway” leading into the flower, and touch-sensitive stigmas, which form lobes that close together after being touched and brushed with pollen, reducing the chances of self-pollination. However, six species of Mimulus are highly self-fertilizing and have cleistogamous or nearly cleistogamous flowers, which are small, closed flowers that do not permit insect pollination.
Phylogenetic analysis shows that these six species do not make up a single subgroup of self-pollinators, but in each case, their most closely related sister taxon is a bee-pollinated species. This type of situation is not uncommon; it occurs in the genus Clarkia (Onagraceae or willow-herb family), for example. Willis focused on an investigation of the genetic changes that occur in the transition from the classic bee flowers of M. guttatus to the self-pollinated cleistogamous flowers of its sister taxon M. nasutus. The group conducted quantitative trait loci (QTL) mapping of seven floral traits: corolla width, corolla length, throat width, and tube length (which are associated with pollinator attraction), and stigma length, anther height, and stigma-anther separation (which are associated with autogamous selfing). They constructed a linkage map with 255 markers on 14 linkage groups (representing the 14 chromosomes) from an interspecific cross of M. guttatus and M. nasutus and found that there were QTLs associated with floral morphology on each of the 14 linkage groups and that each of the seven traits was affected by 11 to 14 QTLs (Fishman et al., 2001).
Further analysis of the F2 hybrid generation suggested that hybrid sterility in this genus is attributable in part to deleterious genetic combinations of different genomic regions from the two species and, possibly, chromosomal distortions affecting loci that influence pollen–pistil interactions (Fishman and Willis, 2001). This work provides a detailed view of the complex genetic changes that occurred during the evolution of self-fertilization and reproductive isolation.
Andrew Stephenson (Penn State University) discussed the mating system from a slightly different perspective, that of variation in the strength of self-incompatibility (SI) systems that may allow for plants to self-fertilize under certain environmental conditions. Stephenson's group quantified variation in SI among individual plants for natural populations of Campanula rapunculoides and Solanum caronlinense and found that self-fertility in both species increases in the absence of cross-pollination.
In greenhouse experiments, they demonstrated that there is heritable genetic variation for the strength of SI in both Campanula and Solanum (Good-Avila and Stephenson, 2002). In field experiments, they found that the self-pollination rate in genetically identical populations of Campanula varied from highly outcrossing to highly self-pollinating when pollinator availability was manipulated experimentally (Good-Avila et al., 2001). High pollinator availability favored the strong SI phenotypes, whereas limited pollinator availability favored the transmission of weak SI phenotypes. These findings suggest that interactions between plant genotype and the environment determine the actual mating system of SI species in natural populations.
The quality of the presentations and animated discussions during the symposium left little doubt that although much remains to be learned, the evolution and genetic control of plant reproduction is perhaps no longer quite the abominable mystery of Darwin's time.
